Rotavirus is the major cause of severe, life-threatening gastroenteritis in young children and animals. Rotaviruses are large (1000 Angstroms), complex, icosahedral assemblies. This virus has been the subject of extensive biochemical, genetic and structural studies because of its medical relevance, intriguing structural complexity, and unique strategies of replication and morphogenesis. Rotaviruses contain 11 segments of double-stranded RNA encapsidated within three concentric capsid layers. Of the 12 proteins encoded by the genome, six are structural (VP 1-7), and six are non-structural (NSP 1-6). In the last four years, we have made exciting new discoveries that have provided a better characterization of the rotavirus structure and a deeper insight into the structural basis of various virus functions such as trypsin-enhanced infectivity, virus assembly, endogenous transcription, and genome replication and packaging. These recent developments, together with other developments in the molecular biology of rotaviruses, have allowed us to plan more in-depth dissection of structure-function correlations in rotavirus using a combination of high-resolution cryo-EM and X-ray crystallographic techniques. The specific objectives of the proposed project are: 1) To further investigate the mechanism of protease-enhanced infectivity and spike assembly, and structural basis of receptor-mediated cell entry of rotavirus. 2) To further our understanding of the structural basis of endogenous transcription in rotavirus by characterizing the structural alterations in response to transcriptional activation using high-resolution cryo-EM techniques. 3) To dissect the structural mechanisms of rotavirus genome replication/packaging using X-ray crystallographic techniques. Our structural information in conjunction with continued advances in the molecular virology of rotavirus would have the potential to enhance the development of more effective methods of disease prevention and control. More importantly, we expect to continue to discover new fundamental structural information to help understand how these complex viruses gain entry into host cells, assemble, transcribe, replicate, and package their genomes.